The long range goal of this project is to determine if airway neurons contribute to the pathogenesis of ozone-induced airways hyperresponsiveness. The proposed studies will focus on the responses of intrinsic airway neurons: those neurons that reside entirely within the airway walls. In specific Aim 1 , communications and projections between intrinsic airway neurons will be defined, testing the hypothesis that intrinsic airway neurons in ferrets form functional and structural interneuronal connections. The interneuronal projections between acetylcholine (Ach)-, substance P (SP), vasoactive intestinal peptide (VIP)- and nitric oxide (NO)-containing airway neurons using neuroanatomical tracing procedures will be characterized. These studies will establish specific neural pathways involved in neural regulation of airway function. Structural contacts between intrinsic airway neurons will be verified using electron microscopy. Specific aims 2 and 3 will test the hypothesis that neurotransmitters synthesized by intrinsic airway neurons is altered by ozone inhalation and that these changes contribute directly the development of airways hyperresponsiveness. Ferrets will be exposed to 1.2 ppm ozone for one hour or 5 days. After 1 hr, 24 hr or 14 days, mediator expression will be evaluated using immunocytochemical and in situ hybridization procedure. Ventilatory changes and cholinergic smooth muscle responses will be measured. In some experiments, the effect of surgical removal of airway neurons on smooth muscle and ventilatory responses will be determined. The proposed studies will provide information critical to understand the organization of neuronal circuits in the airways and their possible involvement in the pathogenesis of ozone-induced airway responses. changes in neurotransmitter expression by airway neurons may be a common pathogenetic mechanism mediating airway hyperresponsiveness in asthma. The results from these studies may provide the scientific basis for developing protective or therapeutic strategies to selectively target airway neurons using inhaled medications.